C1QTNF9 Antibody, FITC conjugated

What is C1QTNF9 and why is it a target of interest in research?

C1QTNF9 (also known as CTRP9) is a member of the C1q/tumor necrosis factor-related protein family that functions as a novel regulator of endothelial progenitor cell (EPC) function. Research has demonstrated that globular C1QTNF9 (gCTRP9) can restore high-glucose-suppressed EPC functions by activating endothelial nitric oxide synthase (eNOS). This protein plays important roles in various physiological processes, including vascular function, inflammation, and metabolism. As a secreted protein, C1QTNF9 represents a significant target for investigating cell signaling pathways, particularly in the context of vascular homeostasis and diabetes-related complications .

What are the primary applications for C1QTNF9 Antibody with FITC conjugation?

The FITC-conjugated C1QTNF9 polyclonal antibody is primarily utilized in fluorescence-based applications including:

• Western Blotting (WB) for protein detection and quantification

• Immunofluorescence on paraffin-embedded tissues (IF/IHC-P)

• Immunofluorescence on frozen tissue sections (IF/IHC-F)

• Immunocytochemistry (ICC) for cellular localization studies

The direct FITC conjugation eliminates the need for secondary antibody incubation, reducing protocol time and potential cross-reactivity issues in multi-labeling experiments.

What species reactivity does the commercially available C1QTNF9 FITC-conjugated antibody demonstrate?

According to product specifications, the bs-15085R-FITC C1QTNF9 polyclonal antibody has confirmed reactivity with rat samples. Additionally, it shows predicted reactivity with human, mouse, dog, and horse samples based on sequence homology analysis . For research requiring cross-species applications, validation testing is recommended before proceeding with full-scale experiments.

What are the recommended working dilutions for different applications using FITC-conjugated C1QTNF9 antibody?

The optimal working dilutions for the FITC-conjugated C1QTNF9 polyclonal antibody vary by application:

• Western Blotting: 1:300-1:5000

• Immunofluorescence (IHC-P): 1:50-1:200

• Immunofluorescence (IHC-F): 1:50-1:200

• Immunocytochemistry (ICC): 1:50-1:200

These ranges provide starting points for optimization. Researchers should perform titration experiments to determine the optimal concentration for their specific sample type and experimental conditions.

How should samples be prepared for optimal detection using C1QTNF9 FITC-conjugated antibody?

Sample preparation depends on the application:

For cellular samples:

• Fix cells with 4% paraformaldehyde for 15 minutes

• Permeabilize if detecting intracellular targets

• Block with appropriate blocking buffer containing 1-5% BSA

• Incubate with the antibody at optimized dilution

• Counterstain nuclei with DAPI if desired

• Mount with anti-fade mounting medium to preserve fluorescence

For tissue sections:

• Use standard deparaffinization and antigen retrieval methods for FFPE tissues

• For frozen sections, fix with acetone or 4% paraformaldehyde

• Block endogenous peroxidases and non-specific binding sites

• Apply the antibody at recommended dilutions

• Wash thoroughly and counterstain as appropriate

What controls should be included when using FITC-conjugated C1QTNF9 antibody?

Essential controls include:

• Positive control: Samples known to express C1QTNF9 (based on literature or previous validation)

• Negative control: Samples known not to express C1QTNF9

• Isotype control: FITC-conjugated rabbit IgG at the same concentration as the primary antibody

• Secondary antibody-only control (for indirect detection methods)

• Unstained sample control to assess autofluorescence

• Blocking peptide control to verify specificity

For quantitative applications, include a standard curve with recombinant C1QTNF9 protein.

How can FITC-conjugated C1QTNF9 antibody be incorporated into multi-color flow cytometry panels?

When designing multi-color flow cytometry panels including FITC-conjugated C1QTNF9 antibody:

• Consider spectral overlap: FITC emits in the green spectrum (~520 nm), so avoid fluorophores with significant spectral overlap such as GFP or PE

• Perform compensation controls for each fluorophore

• Titrate the antibody to determine optimal concentration

• Include FMO (Fluorescence Minus One) controls

• If analyzing cells with high autofluorescence, consider alternative conjugates with emission spectra further from the autofluorescence range

• For intracellular staining, optimize permeabilization protocols to maintain cell integrity while allowing antibody access

What approaches are recommended for studying C1QTNF9 in high-glucose cellular environments?

Based on current research methodologies:

• Cell Culture Setup:

  • Maintain cells in standard growth medium

  • Expose experimental groups to high glucose (25 mmol/L) for established periods (e.g., 4 days)

  • Include osmotic control groups with mannitol (25 mmol/L)

  • Treat with recombinant gCTRP9 (0.5-10 μg/mL) for various time periods (2-24 hours)

• Analytical Methods:

  • Assess cell migration, adhesion, and tube formation capabilities

  • Measure protein expression of adiponectin receptors and N-cadherin using Western blotting

  • Determine phosphorylation states of AMP-activated protein kinase, protein kinase B, and eNOS

  • Quantify nitrite production to assess eNOS activity

  • Use the FITC-conjugated C1QTNF9 antibody to track protein localization under varying glucose conditions

How can C1QTNF9 antibody be used in co-localization studies with other cellular markers?

For effective co-localization studies:

• Select compatible fluorophores for multi-labeling:

  • FITC-conjugated C1QTNF9 antibody (green emission)

  • Combine with red fluorophores (e.g., Texas Red, Cy3) or far-red fluorophores (e.g., Cy5, APC) for other targets

  • Include DAPI nuclear counterstain (blue emission)

• Sequential staining approach:

  • Apply FITC-conjugated C1QTNF9 antibody first

  • Wash thoroughly

  • Apply additional antibodies with compatible host species

  • Use confocal microscopy for high-resolution co-localization analysis

• Signal quantification:

  • Calculate Pearson's or Manders' correlation coefficients

  • Perform intensity correlation analysis

  • Use specialized co-localization software for precise quantification

What are common issues when using FITC-conjugated antibodies and how can they be resolved?

How should researchers quantify and interpret C1QTNF9 expression in fluorescence microscopy experiments?

For accurate quantification and interpretation:

• Image Acquisition:

  • Use consistent exposure settings between samples

  • Capture multiple representative fields (minimum 5-10)

  • Include z-stack images for three-dimensional structures

  • Ensure images are not saturated

• Quantification Methods:

  • Measure mean fluorescence intensity (MFI) of regions of interest

  • Calculate corrected total cell fluorescence (CTCF) = Integrated Density - (Area of selected cell × Mean fluorescence of background)

  • Determine percentage of positive cells in population

  • Assess co-localization coefficients if performing dual-labeling

• Statistical Analysis:

  • Compare treatment groups using appropriate statistical tests

  • Include sufficient biological and technical replicates

  • Report standard deviation or standard error of measurements

  • Normalize to appropriate controls when comparing across experiments

How can researchers overcome tissue autofluorescence when using FITC-conjugated C1QTNF9 antibody?

Strategies to mitigate tissue autofluorescence include:

• Pre-treatment methods:

  • Incubate sections with Sudan Black B (0.1-0.3% in 70% ethanol) for 10-20 minutes

  • Use commercial autofluorescence quenchers specific to tissue type

  • Photobleach samples with light exposure before antibody application

• Acquisition adjustments:

  • Utilize spectral imaging and linear unmixing to separate FITC signal from autofluorescence

  • Apply bandpass filters with narrow wavelength ranges

  • Use time-gated detection if available

• Post-acquisition processing:

  • Subtract autofluorescence using unstained control images

  • Apply computational algorithms for autofluorescence removal

  • Consider alternative fluorophores with emission in ranges less affected by autofluorescence

What are the advantages of FITC-conjugated C1QTNF9 antibody compared to unconjugated primary antibodies?

The FITC-conjugated C1QTNF9 antibody offers several advantages over unconjugated alternatives:

• Reduced protocol time: Eliminates secondary antibody incubation and washing steps

• Decreased non-specific binding: Avoids potential cross-reactivity of secondary antibodies

• Direct quantification: Signal intensity directly correlates with antigen abundance

• Multiplexing capability: Allows simultaneous detection of multiple targets when combined with antibodies raised in the same host species but conjugated to different fluorophores

• Increased sensitivity: Direct conjugation can reduce signal loss that occurs during secondary detection steps

How do results from fluorescence-based detection compare with ELISA for C1QTNF9 quantification?

Comparing fluorescence-based and ELISA detection methods:

What novel research applications could benefit from using FITC-conjugated C1QTNF9 antibody?

Emerging research applications include:

• Live-cell imaging of C1QTNF9 trafficking and secretion:

  • Real-time visualization of protein movement

  • Kinetic studies of secretion in response to stimuli

  • Internalization and receptor interactions

• High-content screening applications:

  • Drug discovery targeting C1QTNF9 pathways

  • Phenotypic screening of cellular responses

  • Automated analysis of large sample sets

• Extracellular vesicle (EV) characterization:

  • Detection of C1QTNF9 in EVs using flow cytometry

  • Sorting of C1QTNF9-positive vesicle populations

  • Tracking EV uptake and content delivery

• Single-cell analysis techniques:

  • Flow cytometry-based sorting of C1QTNF9-expressing populations

  • Correlation with other cellular markers

  • Integration with transcriptomic and proteomic approaches

• In vivo imaging applications:

  • Tracking of labeled cells in animal models

  • Evaluation of tissue distribution and clearance

  • Assessment of therapeutic interventions targeting C1QTNF9 pathways